No abstract

The circadian clock times cellular processes to the day/night cycle via a Transcription-Translation negative Feedback Loop (TTFL). However, a mechanistic understanding of the negative arm in both the timing of the TTFL and its control of output is lacking. We posited that the formation of negative-arm protein complexes was fundamental to clock regulation stemming from the negative arm. Using a modified peptide microarray approach termed Linear motif discovery using rational design (LOCATE), we characterized the interaction of the disordered negative-arm clock protein FREQUENCY to its partner protein FREQUENCY-Interacting RNA helicase. LOCATE identified a specific Short Linear Motif (SLiM) and interaction hotspot as well as positively charged islands that mediate electrostatic interactions, suggesting a model where negative arm proteins form a fuzzy complex essential for clock timing and robustness. Further analysis revealed that the positively charged islands were an evolutionarily conserved feature in higher eukaryotes and contributed to proper clock function.

Horizontal gene transfer via plasmids could favour cooperation in bacteria, because transfer of a cooperative gene turns non-cooperative cheats into cooperators. This hypothesis has received support from both theoretical and genomic analyses. In contrast, with a comparative analysis across 51 diverse species, we found that genes for extracellular proteins, which are likely to act as cooperative ‘public goods’, were not more likely to be carried on either: (i) plasmids compared to chromosomes; or (ii) plasmids that transfer at higher rates. Our results were supported by theoretical modelling which showed that while horizontal gene transfer can help cooperative genes initially invade a population, it does not favour the longer-term maintenance of cooperation. Instead, we found that genes for extracellular proteins were more likely to be on plasmids when they coded for pathogenic virulence traits, in pathogenic bacteria with a broad host-range. Taken together, these results support an alternate hypothesis, that plasmid gene location confers benefits other than horizontal gene transfer.

Bacteriocins are antimicrobial toxins produced by bacteria to defend and invade territories by killing unrelated strains and species. Understanding if bacteriocins shape natural populations is important for understanding the evolution of antimicrobial resistance and identifying novel antimicrobials for clinical use. Staphylococcus aureus is an opportunistic pathogen that asymptomatically colonises the nasal cavity of 1 in 3 healthy adults. S. aureus is known to produce many different bacteriocins, however we are yet to understand the extent to which they mediate the establishment of nasal populations. Here, we test the importance of bacteriocins in driving colonisation success, by screening S. aureus antimicrobial inhibition against otherS. aureus strains and three commensal species that commonly co-inhabit the nasal cavity. We use a longitudinally sampled collection of 173S. aureusnasal isolates from 14 participants over 90-months to track within-individual population changes over time. We found that 8% of allS. aureus isolates produced bacteriocins active against other species, but that between-strain bacteriocin inhibition inS. aureus was very rare, observed by only 0.5% of isolates. Therefore, while there is no evidence that intraspecific competition drives colonisation success in the nasal cavity, interspecific competition is more likely to influence strain and species dominance.

Many bacterial genes encode proteins that are secreted extracellularly. These proteins can be considered cooperative because all surrounding cells can benefit from their production. Therefore, it has been hypothesized that these cooperative genes would more frequently lie on mobile elements, such as plasmids, which can transfer to other cells. This could stabilise cooperation, leading to the prediction that plasmids should carry proportionally more cooperative genes than the less mobile chromosome. However, it is unknown whether this prediction holds across the bacterial tree of life. To address this, we analysed the gene content of the chromosome and plasmid(s) of 1620 genomes comprising 51 diverse bacterial species. We find that across species analysed, plasmids do not carry proportionally more cooperative genes than the chromosome. Contrary to prediction, the role of mobile elements in promoting cooperative behaviour is highly variable across bacterial species.